NF-kB transcription factors are known to regulate the expression of a number of genes involved in T cell activation and function. Some evidence has suggested that they also play a role in T cell development. However, the role of the NF-kB in Ag-induced thymocyte differentiation has not been directly addressed to date. We critically examined this role by employing DPK, a CD4+CD8+ thymocyte line that undergoes differentiation upon TCR engagement in a process that closely mimics positive selection. Expression of a degradation-resistant form of IkBa in DPK cells resulted in constitutive inhibition of NF-kB activity. We found that in the absence of NF-kB activity, MHC-peptide-induced differentiation of DPK was blocked. Furthermore, differentiation induced by a nonphysiologic stimulus, anti-TCR Ab, was greatly reduced. Altogether, our data indicate a requirement for NF-kB in the developmental changes associated with positive selection. Also, as a model system for the regulation of cell differentiation by DNA binding factors, we are studying the role C/EBP and its associated molecules in this process. Moreover, the b1 integrin gene has been knocked out through traditional means by two research groups and in each case has been shown to be embryonic lethal. We have cloned an appropriate b1 genomic fragment and engineered it for tissue-specific (thymus) knockout production. By deleting the b1 gene only from thymocytes, we expect to be able to study its impact on the in vivo development of the immune system.